Fungal natural products are complex molecules containing a wide range of chemical motifs. Many of these natural products are involved in pathogenesis, and many have pharmacological activity. This proposal focuses on the biosynthetic pathway of one natural product, equisetin. The goal of the research is to gather specific information that will be useful in efforts to make precise alterations to the biosynthetic pathway that will result in alterations to the structure of equisetin. Like many other related molecules, equisetin is toxic and interacts specifically with an important drug target (HIV-1 integrase, in the case of equisetin). The ability to alter the structures of equisetin and related molecules would assist in making drug candidates that are less generally toxic or are more effective against specific targets. The category of fungal natural products that includes equisetin is produced by hybrid polyketide synthase/non-ribosomal peptide synthase (PKS-NRPS) enzymes. These large enzymes contain many domains. Two of the domains are used to attach the growing equisetin molecule to the enzyme during its synthesis. Each other domain catalyzes a specific reaction in the synthesis of the equisetin molecule. PKS-NRPS products are made up of a polyketide (produced by the PKS) to which the NRPS adds an amino acid. The NRPS consists of four domains. One domain (T) is a carrier for the equisetin molecule, a second domain (A) is required for activating the amino acid substrate, a third (C) catalyzes the attachment of the amino acid to the polyketide, and the last domain (R) catalyzes the release of the product from the carrier domain. My specific aims are 1) to test the role of each equisetin synthase NRPS domain in determining which amino acid is used as a substrate, 2) to identify regions and specific residues in the domains that can be mutated to alter substrate specificity and 3) to test whether other fungal PKS-NRPS enzymes' R domains catalyze product release by different mechanisms. The experimental approach will involve expression and purification of the four equisetin synthase NRPS domains along with the carrier domain from the PKS, and R domains from two other PKS-NRPS enzymes. In the first specific aim, I will learn about the amino acid substrate specificities of the different domains, with the goal of understanding which parts of the whole PKS-NRPS are responsible for favoring the particular amino acid that is incorporated into equisetin, and whether different amino acids could be incorporated as well. In the second part of the research, I will create hybrid NRPS enzymes with domains from equisetin synthase and a closely related NRPS, and use random and directed mutagenesis to improve the activities of these hybrid enzymes. In the third specific aim, I will test R domains from other PKS-NRPS enzymes for a release mechanism different from the mechanism used by the equisetin synthase R domain, and will combine the catalytic activities of R domains from different synthases with the EqiS condensation and adenylation domains.